Regulation of glycoprotein Ib-IX-von Willebrand factor interaction by cAMP-dependent protein kinase-mediated phosphorylation at Ser166 of glycoprotein Ibβ
Regulation of glycoprotein Ib-IX-von Willebrand factor interaction by cAMP-dependent protein kinase-mediated phosphorylation at Ser166 of glycoprotein Ibβ
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DOI：
10.1074/jbc.m208329200
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发表时间：
2002-12-06
影响因子：
4.8
通讯作者：
Du, XP
中科院分区：
文献类型：
--
作者：
Bodnar, RJ;Xi, XD;Du, XP
血管性血友病因子(VWF)的血小板受体，即糖蛋白(GP)Ib-IX，介导了最初的血小板黏附和激活。已知GPIbbeta的胞质结构域在Ser(166)处被cAMP依赖的蛋白激酶(PKA)磷酸化。为了了解GPIbbeta磷酸化的生理作用，构建了一个用丙氨酸取代GPIbbeta的Ser(166)的突变体(S166A)和一个缺失GPIbbeta的166-181残基的缺失突变体(Delta165)。这些突变体在中国仓鼠卵巢(CHO)细胞中表达，与野生型GPIb-IX相比，显示出更强的VWF结合功能。用PKA抑制剂PKI处理表达野生型GPIB-IX的CHO细胞，降低了Ser(166)的磷酸化，也增强了VWF与GPIB-IX的结合。此外，在流动条件下，表达S166A或Delta165突变体的细胞对固定化VWF的粘附性显著增强。与在CHO细胞中的研究一致，用PKI处理血小板可增强VWF与血小板的结合。相反，PKA刺激剂Forsklin减少了VWF的结合和VWF诱导的血小板凝集，这一作用可被PKI逆转。因此，PKA介导的GPIbβ在Ser(166)处的磷酸化负向调节VWF与GPIb-IX的结合，是PKA介导血小板抑制的机制之一。
The platelet receptor for von Willebrand factor (VWF), glycoprotein (GP) Ib-IX, mediates initial platelet adhesion and activation. It is known that the cytoplasmic domain of GPIbbeta is phosphorylated at Ser(166) by cAMP-dependent protein kinase (PKA). To understand the physiological role of GPIbbeta phosphorylation, a GPIb-IX mutant replacing Ser(166) of GPIbbeta with alanine (S166A) and a deletion mutant lacking residues 166-181 of GPIbbeta (Delta165) were constructed. These mutants, expressed in Chinese hamster ovary (CHO) cells, showed an enhanced VWF-binding function compared with wild type GPIb-IX. Treatment of CHO cells expressing wild type GPIb-IX with a PKA inhibitor, PKI, reduced Ser(166) phosphorylation and also enhanced VWF binding to GPIb-IX. Furthermore, cells expressing S166A or Delta165 mutants showed a significantly enhanced adhesion to immobilized VWF under flow conditions. Consistent with the studies in CHO cells, treatment of platelets with PKI enhanced VWF binding to platelets. In contrast, a PKA stimulator, forskolin, reduced VWF binding and VWF-induced platelet agglutination, which was reversed by PKI. Thus, PKA-mediated phosphorylation of GPIbbeta at Ser(166) negatively regulates VWF binding to GPIb-IX and is one of the mechanisms by which PKA mediates platelet inhibition.